Methods for cloning animals

ABSTRACT

The present invention pertains to methods for cloning animals. In particular, the invention includes methods of cloning an animal by combining a genome from an activated donor cell with an activated enucleated oocyte to thereby obtain a nuclear transfer embryo, and impregnating an animal with the nuclear transfer embryo in conditions suitable for gestation of a cloned animal. The invention further relates to methods of chemically enucleating an oocyte having a meiotic spindle apparatus by exposing the oocyte with a compound that destabilizes the meiotic spindle apparatus.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/432,906, filed Nov. 2, 1999, which claims the benefit of priority toU.S. Provisional Application No. 60/149,317, filed on Aug. 17, 1999,entitled, “Induced Enucleation Methods To Clone Non-Human Animals,” byBaguisi et al.; U.S. Provisional Application No. 60/131,061, filed onApr. 26, 1999, entitled, “Use of Telophase Oocytes to Clone Non-HumanAnimals,” by Baguisi et al; U.S. Provisional Application No. 60/131,328,filed Apr. 26, 1999, entitled, “Transgenic and Cloned Mammals,” byBaguisi, et al.; and U.S. Provisional Application No. 60/106,728, filedNov. 2, 1998, entitled, “Transgenic and Cloned Mammals,” by Echelard, etal.

GOVERNMENT SUPPORT

This invention was made with Government support under GM35395, awardedby the National Institutes of Health. The Government has certain rightsin the invention.

The entire teachings of the above applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

The production of desired proteins is useful in drug development andtreatment of diseases. Several traditional methods for producingproteins, especially in high volume, are often inadequate for severalreasons. Transgenic technology or cloning technology can lead to severaladvancements in medicine, including the production of useful proteins.Transgenic or cloning technology allows for the introduction of atransgenic nucleotide sequence into a host animal, thereby allowing forthe expression of this transgenic nucleotide sequence, and production ofthe protein.

Accordingly, few reliable methods exist for producing transgenic orcloned animals, especially those methods that are able to produce usefulproteins. Hence, a need exists for producing transgenic or clonedanimals, and in particular, animals that make such desirable proteins.

SUMMARY OF THE INVENTION

The present invention provides effective methods for producingtransgenic or cloned animals, and for obtaining useful proteins. Theinvention includes methods for cloning an animal by combining a genomefrom an activated donor cell with an activated, enucleated oocyte tothereby form a nuclear transfer embryo, and impregnating an animal withthe nuclear transfer embryo in conditions suitable for gestation of thecloned animal. The activated donor cell is in a stage of the mitoticcell cycle such as G₁ phase, S phase, or G₂/M phase. The activated donorcell can be a variety of cells such as a somatic cell (e.g., an adultsomatic cell or an embryonic somatic cell), a germ cell or a stem cell.Types of somatic cells include fibroblast cells or epithelial cells. Theactivated, enucleated oocyte is in a stage of the meiotic cell cycle,such as metaphase I, anaphase I, anaphase II or telophase II. The oocytecan be enucleated chemically, by X-ray irradiation, by laser irradiationor by physical removal of the nucleus.

The invention also includes a method of producing a transgenic animal bycombining a genetically engineered genome from an activated donor cellwith an activated, enucleated oocyte to thereby form a transgenicnuclear transfer embryo; and impregnating an animal with the transgenicnuclear transfer embryo in conditions suitable for gestation of thetransgenic animal. The stages of the cell cycle for the activated donorcell and the activated, enucleated oocyte are described above. The typesof activated donor cell are also described above. The oocyte can beenucleated chemically, by X-ray irradiation, by laser irradiation or byphysical removal of the nucleus.

The present invention also relates to methods of producing a nucleartransfer embryo, comprising combining a genome from an activated donorcell with an activated, enucleated oocyte. The oocyte is activated byexposing the oocyte to increased levels of calcium, and/or decreasingphosphorylation in the oocyte. Compounds or conditions that activate theoocyte are, for example, ethanol, ionophore or electrical stimulation inthe presence of calcium. Increases of calcium can be between above 10%and 60% above baseline levels of calcium. The donor cell is activated byreducing the nutrients in the serum of the donor cell (e.g., 0.5% FetalBovine Serum) for a period of time, and then exposing the donor cell toserum having an increased amount of nutrients (10% Fetal Bovine Serum).Combining a genome from an activated donor cell with an activated oocytecan include fusing the activated donor cell with the activated oocyte,or microinjecting the nucleus of the activated donor cell into theactivated oocyte.

The present invention also pertains to methods of producing a protein ofinterest in an animal, comprising combining a genome from an activateddonor cell with an activated, enucleated oocyte to thereby form anuclear transfer embryo, wherein the genome from the activated donorcell encodes the protein of interest; impregnating an animal with thenuclear transfer embryo in conditions suitable for gestation of a clonedanimal; and purifying the protein of interest from the cloned animal.Purification of the protein of interest can be expressed in tissue,cells or bodily secretion of the cloned animal. Examples of such tissue,cells or bodily secretions are milk, blood, urine, hair, mammary gland,muscle, viscera (e.g., brain, heart, lung, kidney, pancreas, gallbladder, liver, stomach, eye, colon, small intestine, bladder, uterusand testes).

The present invention further encompasses a method of producing aheterologous protein in a transgenic animal comprising combining agenetically engineered genome from an activated donor cell with anactivated, enucleated oocyte to thereby form a nuclear transfer embryo,wherein the genome from the activated donor cell encodes theheterologous protein; impregnating an animal with the nuclear transferembryo in conditions suitable for gestation of the nuclear transferembryo into a cloned animal; and recovering the heterologous proteinfrom the cloned animal. The genetically engineered genome includes anoperatively linked promoter (e.g., a host endogenous promoter, anexogenous promoter and a tissue-specific promoter). Examples oftissue-specific promoters are mammary-specific promoter, blood-specificpromoter, muscle-specific promoter, neural-specific promoter,skin-specific promoter, hair-specific promoter and urinary-specificpromoter.

The present invention also embodies methods of enucleating an oocytehaving a meiotic spindle apparatus, by exposing the oocyte with acompound that destabilizes the meiotic spindle apparatus. Destabilizingthe meiotic spindle apparatus results in destabilizing microtubules,chromosomes, or centrioles. Compounds that can destabilize the meioticspindle apparatus are, for example, demecolcine, nocodazole, colchicine,and paclitaxel. To further enhance destabilization of the mieoticspindle apparatus, the temperature, osmolality or composition of mediumwhich surrounds the oocyte can be altered.

Additionally, the invention includes methods of preparing an oocyte fornuclear transfer, comprising: exposing the oocyte to ethanol, ionophore,or to electrical stimulation, to thereby obtain an activated oocyte, andsubjecting the activated oocyte to a compound that destabilizes meioticspindle apparatus, to thereby enucleate the activated oocyte. Thecompounds described above destabilize the meiotic spindle apparatus. Theactivated oocyte can be in a stage of a meiotic cell cycle, such asmetaphase I, anaphase I, anaphase II and telophase II.

The present invention advantageously allows for more efficient cloningmethods. By fusing or combining an activated oocyte with the genome froman activated donor cell, the resulting nuclear transfer embryo is morecompetent to develop. This developmentally competent nuclear transferembryo results in improved pregnancy rates of an animal impregnated withthe nuclear transfer embryo. These animals give birth to cloned animals.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of cloning an animal bycombining an activated oocyte with the genome from an activated donorcell. “Cloning an animal” refers to producing an animal that developsfrom an oocyte containing genetic information or the nucleic acidsequence of another animal, the animal being cloned. The cloned animalhas substantially the same or identical genetic information as that ofthe animal being cloned. “Cloning” also refers to cloning a cell, whichincludes producing an oocyte containing genetic information or thenucleic acid sequence of another animal. The resulting oocyte having thedonor genome is referred to herein as a “nuclear transfer embryo.”

The present invention encompasses the cloning of a variety of animals.These animals include mammals (e.g., human, canines, felines), murinespecies (e.g., mice, rats), and ruminants (e.g., cows, sheep, goats,camels, pigs, oxen, horses, llamas). In particular, goats of Swissorigin, for example, the Alpine, Saanen and Toggenburg bread goats, wereused in the Examples described herein. The donor cell and the oocyte arepreferably from the same animal.

Both the donor cell and the oocyte must be activated. An activated(e.g., non-quiescent) donor cell is a cell that is in actively dividing(e.g., not in a resting stage of mitosis). In particular, an activateddonor cell is one that is engaged in the mitotic cell cycle, such as G₁phase, S phase or G₂/M phase. The mitotic cell cycle has the followingphases, G₁, S, G₂ and M. The G₂/M phase refers to the transitional phasebetween the G₂ phase and M phase. The commitment event in the cellcycle, called START (or restriction point), takes place during the G₁phase. “START” as used herein refers to late G₁ stage of the cell cycleprior to the commitment of a cell proceeding through the cell cycle. Thedecision as to whether the cell will undergo another cell cycle is madeat START. Once the cell has passed through START, it passes through theremainder of the G₁ phase (i.e., the pre-DNA synthesis stage). The Sphase is the DNA synthesis stage, which is followed by the G₂ phase, thestage between synthesis and mitosis. Mitosis takes place during the Mphase. If prior to START, the cell does not undergo another cell cycle,the cell becomes arrested. In addition, a cell can be induced to exitthe cell cycle and become quiescent or inactive. A “quiescent” or“inactive” cell, is referred to as a cell in G₀ phase. A quiescent cellis one that is not in any of the above-mentioned phases of the cellcycle. Preferably, the invention utilizes a donor cell is a cell in theG₁ phase of the mitotic cell cycle.

It is preferable that the donor cells be synchronized. Using donor cellsat certain phases of the cell cycle, for example, G₁ phase, allows forsynchronization of the donor cells. One can synchronize the donor cellsby depriving (e.g., reducing) the donor cells of a sufficient amount ofnutrients in the media that allows them to divide. Once the donor cellshave stopped dividing, then the donor cells are exposed to media (serum)containing a sufficient amount of nutrients to allow them to beingdividing (e.g., mitosis). The donor cells begin mitosis substantially atthe same time, and are therefore, synchronous. For example, the donorcells are deprived of a sufficient concentration of serum by placing thecells in 0.5% Fetal Bovine Serum (FBS) for about a week. Thereafter, thecells are placed in about 10% FBS and they will begin dividing at aboutthe same time. They will enter the G1 phase about the same time, and aretherefore, ready for the cloning process. See the Exemplificationsection for details about the synchronization of the donor cells.

Methods of determining which phase of the cell cycle a cell is in areknown to those skilled in the art, for example, U.S. Pat. No. 5,843,705to DiTullio et al., Campbell, K. H. S., et al., Embryo TransferNewsletter, vol. 14(1):12-16 (1996), Campbell, K. H. S., et al., Nature,380: 64-66 (1996), Cibelli, J. B., et al., Science, 280: 1256-1258(1998), Yong, Z. and L. Yuqiang, Biol. of Reprod., 58: 266-269 (1998)and Wilmut, I., et al., Nature, 385: 810-813 (1997). For example, asdescribed below in the Examples, various markers are present atdifferent stages of the cell cycle. Such markers can include cyclines D1, 2, 3 and proliferating cell nuclear antigen (PCNA) for G₁, and BrDuto detect DNA synthetic activity. In addition, cells can be induced toenter the G₀ stage by culturing the cells on a serum-deprived medium.Alternatively, cells in G₀ stage can be induced to enter into the cellcycle, that is, at G₁ stage by serum activation (e.g., exposing thecells to serum after the cells have been deprived of a certain amount ofserum).

The donor cell can be any type of cell that contains a genome or geneticmaterial (e.g., nucleic acid), such as a somatic cell, germ cell or astem cell. The term “somatic cell” as used herein refers to adifferentiated cell. The cell can be a somatic cell or a cell that iscommitted to a somatic cell lineage. Alternatively, any of the methodsdescribed herein can utilize a diploid stem cell that gives rise to agerm cell in order to supply the genome for producing a nuclear transferembryo. The somatic cell can originate from an animal or from a celland/or tissue culture system. If taken from an animal, the animal can beat any stage of development, for example, an embryo, a fetus or anadult. Additionally, the present invention can utilize embryonic somaticcells. Embryonic cells can include embryonic stem cells as well asembryonic cells committed to a somatic cell lineage. Such cells can beobtained from the endoderm, mesoderm or ectoderm of the embryo.Embryonic cells committed to a somatic cell lineage refer to cellsisolated on or after approximately day ten of embryogenesis. However,cells can be obtained prior to day ten of embryogenesis. If a cell lineis used as a source for a chromosomal genome, then primary cells arepreferred. The term “primary cell line” as used herein includes primarycells as well as primary derived cell lines.

Suitable somatic cells include fibroblasts (for example, primaryfibroblasts), epithelial cells, muscle cells, cumulous cells, neuralcells, and mammary cells. Other suitable cells include hepatocytes andpancreatic islets.

The genome of the somatic cell can be the naturally occurring genome,for example, for the production of cloned animals, or the genome can begenetically altered to comprise a transgenic sequence, for example, forthe production of transgenic cloned animals, as further describedherein.

Somatic cells can be obtained by, for example, disassociation of tissueby mechanical (e.g., chopping, mincing) or enzymatic means (e.g.,trypsinization) to obtain a cell suspension followed by culturing thecells until a confluent monolayer is obtained. The somatic cells canthen be harvested and prepared for cryopreservation, or maintained as astalk culture. The isolation of somatic cells, for example, fibroblasts,is described herein.

The oocytes used in the present invention are activated oocytes.Activated oocytes are those that are in a dividing stage of meiotic celldivision, and include metaphase I, anaphase I, anaphase II, andpreferably, telophase II. Oocytes in metaphase II are considered to bein a resting state. The oocytes can be in the resting stage of metaphaseII, and then activated, using methods described herein. The stage thatthe oocyte is in can be identified by visual inspection of the oocyteunder a sufficient magnification. Oocytes that are in telophase II areidentified, for example, by the presence of a protrusion of the plasmamembrane of a second polar body. Methods for identifying the stage ofmeiotic cell division are known in the art.

Oocytes are activated by increasing their exposure to calcium levels.Increasing levels of calcium, e.g., by between about 10% and about 60%above the baseline levels, induces activation or meiotic cell divisionof the oocyte. Baseline levels are those levels of calcium found in aninactive oocyte. Rising levels of calcium, coupled with decreasinglevels of phosphorylation further facilitates activation of the oocyte.Several methods exist that allow for activation of the oocyte. Inparticular, a calcium ionophore (e.g., ionomycin) is an agent thatincreases the permeability of the oocyte's membrane and allows calciumto enter into the oocyte. As the free calcium concentration in the cellincreases during exposure to the ionophore, the oocyte is activatedfollowing reduction in MPF (maturation promoting factor) activity. Suchmethods of activation are described in U.S. Pat. No. 5,496,720. Ethanolhas a similar affect. Prior to or following enucleation, an oocyte inmetaphase II can be activated with ethanol according to the ethanolactivation treatment as described in Presicce and Yang, Mol. Reprod.Dev., 37: 61-68 (1994); and Bordignon & Smith, Mol. Reprod. Dev., 49:29-36 (1998). Exposure of calcium to the oocyte also occurs throughelectrical stimulation. The electrical stimulation allows increasinglevels of calcium to enter the oocyte.

Oocytes can be obtained from a donor animal during that animal'sreproductive cycle. For example, oocytes can be aspirated from folliclesof ovaries at given times during the reproductive cycle (exogenoushormone-stimulated or non-stimulated). Also at given times followingovulation, a significant percentage of the oocytes, for example, are intelophase. Additionally, oocytes can be obtained and then induced tomature in vitro to arrested metaphase II stage. Arrested metaphase IIoocytes, produced in vivo or in vitro, can then be induced in vitro toenter telophase. Thus, oocytes in telophase can readily be obtained foruse in the present invention. In particular, oocytes can be collectedfrom a female animal following super ovulations. Oocytes can berecovered surgically by flushing the oocytes from the oviduct of afemale donor. Methods of inducing super ovulations in, for example,goats and the collection of the oocytes are described herein.

Preferably, the cell stage of the activated oocytes correlates to thestage of the cell cycle of the activated donor cell. This correlationbetween the meiotic stage of the oocyte and the mitotic stage of thedonor cell is also referred to herein as “synchronization.” For example,an oocyte in telophase fused with the genome of a donor cell in G₁ priorto START, provides a synchronization between the oocyte and the donornuclei in the absence of premature chromatin condensation (PCC) andnuclear envelope breakdown (NEBD).

The present invention utilizes an oocyte that is enucleated. Anenucleated oocyte is one that is devoid of the genome, or one that is“functionally enucleated.” A functionally enucleated oocyte contains agenome that is non-functional, e.g., cannot replicate or synthesize DNA.See, for example, Bordignon, V. and L. C. Smith, Molec. Reprod. Dev.,49: 29-36 (1998). Preferably, the genome of the oocyte is removed fromthe oocyte. A genome can be functionally enucleated from the oocyte byirradiation, by x-ray irradiation, by laser irradiation, by physicallyremoving the genome, or by chemical means. Other known methods ofenucleation can be used with the present invention to enucleate theoocyte.

The oocyte can also be rendered functionally inactive by, for example,irradiating the endogenous nuclear material in the oocyte. Methods ofusing irradiation are known to those in the art and are described, forexample, in Bradshaw et al., Molecul. Reprod. Dev., 41: 503-512 (1995).

To physically remove the genome of an oocyte, one can insert amicropipette or needle into the zona pellicuda of the oocyte to removenuclear material from the oocyte. In one example, telophase oocyteswhich have two polar bodies can be enucleated with a micropipette orneedle by removing the second polar body in surrounding cytoplasm.Specifically, oocytes in telophase stage of meiosis can be enucleated atany point from the presence of a protrusion in the plasma membrane fromthe second polar body up to the formation of the second polar bodyitself. Thus, as used herein, oocytes which demonstrate a protrusion inthe plasma membrane, usually with a spindle abutted to it, up toextrusion of a second polar body are considered to be oocytes intelophase. Methods of enucleating a oocyte are described in furtherdetail in the Exemplification Section.

The oocyte can be rendered functionally inactive also by chemicalmethods. Methods of chemically inactivating the DNA are known to thoseof skill in the art. For example, chemical inactivation can be preformedusing the etopsoide-cycloheximide method as described in Fulka andMoore, Molecul. Reprod. Dev., 34: 427-430 (1993). The present inventionincludes enucleating the genome of an oocyte by treating the oocyte witha compound that will induce the oocyte genome (e.g., nuclear chromatin)to segregate into the polar bodies during meiotic maturaton therebyleaving the oocyte devoid of a functional genome, and resulting in theformation of a recipient cytoplast for use in nuclear transferprocedures. Examples of agents that will effect such differentialsegregation include agents that will disrupt 1) cytoskeletal structuresincluding, but not limited to, Taxol® (e.g., paclitaxel), demecolcine,phalloidin, colchicine, nocodozole, and 2) metabolism including, but notlimited to, cycloheximide and tunicamycin. In addition, exposure ofoocytes to other agents or conditions (e.g. increased or decreasedtemperature, pH, osmolality) that preferentially induce the skewedsegregation of the oocyte genome so as to be extruded from the confinesof the oocyte (e.g., in polar bodies) also are included in the preferredmethod. See, for example, methods to include changes in the cytoskeletonand metabolism of cells, methods that are known to those in the artAndreau, J. M. and Timasheff, S. N., Proc. Nat. Acad. Sci. 79: 6753(1982), Obrig, T. G., et al, J. Biol. Chem. 246: 174 (1971), Duskin, D.and Mahoney, W. C., J. Biol. Chem. 257: 3105 (1982), Scialli, A. R., etal, Teratogen, Carcinogen, Mutagen 14: 23 (1994), Nishiyama, I andFujii, T., Exp. Cell Res. 198: 214 (1992), Small, J. V., et al, J. CellSci. 89: 21 (1988), Lee, J. C., et al, Biochem. 19: 6209 (1980).

Combination of the activated, enucleated oocyte and the genome from theactivated donor cell can occur a variety of ways to form the nucleartransfer embryo. A genome of an activated donor cell can be injectedinto the activated oocyte by employing a microinjector (i.e.,micropipette or needle). The nuclear genome of the activated donor cell,for example, a somatic cell, is extracted using a micropipette orneedle. Once extracted, the donor's nuclear genome can then be placedinto the activated oocyte by inserting the micropipette, or needle, intothe oocyte and releasing the nuclear genome of the donor's cell.McGrath, J. and D. Solter, Science, 226: 1317-1319 (1984).

The present invention also includes combining the genome of an activateddonor cell with an activated oocyte by fusion e.g., electrofusion, viralfusion, liposomal fusion, biochemical reagent fusion (e.g.,phytohemaglutinin (PHA) protein), or chemical fusion (e.g., polyethyleneglycol (PEG) or ethanol). The nucleus of the donor cell can be depositedwithin the zona pelliduca which contains the oocyte. The steps of fusingthe nucleus with the oocyte can then be performed by applying anelectric field which will also result in a second activation of theoocyte. The telophase oocytes used are already activated, hence anyactivation subsequent to or simultaneous with the introduction of genomefrom a somatic cell would be considered a second activation event. Withrespect to electrofusion, chambers, such as the BTX® 200Embryomanipulation System for carrying out electrofusion arecommercially available from for example BTX®, San Diego. The combinationof the genome of the activated donor cell with the activated oocyteresults in a nuclear transfer embryo.

A nuclear transfer embryo of the present invention is then transferredinto a recipient animal female and allowed to develop or gestate into acloned or transgenic animal. Conditions suitable for gestation are thoseconditions that allow for the embryo to develop and mature into a fetus,and eventually into a live animal. Such conditions are further describedin the Exemplification Section, and are known in the art. For example,the nuclear transfer embryo can be transferred via the fimbria into theoviductal lumen of each recipient animal female as described in theExemplification Section. In addition, methods of transferring an embryoto a recipient known to those skilled in the art and are described inEbert et al, Bio/Technology, 12: 699 (1994). The nuclear transfer embryocan be maintained in a culture system until at least first cleavage(2-cell stage) up to the blastocyst stage, preferably the embryos aretransferred at the 2-cell or 4-cell stage. Various culture media forembryo development are known to those skilled in the art. For example,the nuclear transfer embryo can be co-cultured with oviductal epithelialcell monolayer derived from the type of animal to be provided by thepractitioner. For example methods of obtaining goat oviductal epithelialcells (GOEC) maintaining the cells in a co-culture are described in theExamples below.

The present invention also relates to methods for generating transgenicanimals by combining an activated oocyte with and a geneticallyengineered genome from an activated donor cell. Such a combinationresults in a transgenic nuclear transfer embryo. A transgenic animal isan animal that has been produced from a genome from a donor cell thathas been genetically altered, for example, to produce a particularprotein (a desired protein). Methods for introducing DNA constructs intothe germ line of an animal to make a transgenic animal are known in theart. For example, one or several copies of the construct may beincorporated into the genome of a animal embryo by standard transgenictechniques.

Embryonal target cells at various developmental stages can be used tointroduce transgenes. A transgene is a gene that produces the desiredprotein and is eventually incorporated into the genome of the activatedoocyte. Different methods are used depending upon the stage ofdevelopment of the embryonal target cell. The specific line(s) of anyanimal used to practice this invention are selected for general goodhealth, good embryo yields, good pronuclear visibility in the embryo,and good reproductive fitness. In addition, the haplotype is asignificant factor.

Genetically engineered donor cells for use in the instant invention canbe obtained from a cell line into which a nucleic acid of interest, forexample, a nucleic acid which encodes a protein, has been introduced.

A construct can be introduced into a cell via conventionaltransformation or transfection techniques. As used herein, the terms“transfection” and “transformation” include a variety of techniques forintroducing a transgenic sequence into a host cell, including calciumphosphate or calcium chloride co-precipitation, DEAE dextrane-mediatedtransfection, lipofection, or electroporation. In addition, biologicalvectors, for example, viral vectors can be used as described below.Samples of methods for transforming or transfecting host cells can befound in Sambrook et al., Molecular Cloning: A Laboratory Manual InSecond Edition, Cold Spring Harbor Laboratory, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. 1989). Two useful andpractical approaches for introducing genetic material into a cell areelectroporation and lipofection.

The DNA construct can be stably introduced into a donor cell line byelectroporation using the following protocol: donor cells, for example,embryonic fibroblasts, are resuspended in phosphate buffer saline (PBS)at about 4×10⁶ cells per mL. Fifty micrograms of linearized DNA is addedto the 0.5 mL cell suspension, and the suspension is placed in a 0.4 cmelectrode gap cuvette. Electroporation is performed using a BioRad GenePulser (Bio Rad) electroporator with a 330 volt pulse at 25 mA, 1000microFarad and infinite resistance. If the DNA construct contains aneomyocin resistance gene for selection, neomyocin resistant clones areselected following incubation where 350 mg/mL of G418 (GIBCO BRL) forfifteen days.

The DNA construct can be stably introduced into a donor somatic cellline by lipofection using a protocol such as the following: about 2×10⁵cells are plated into a 3.5 cm well and transfected with 2 mg oflinearized DNA using LipfectAMINE® (GIBCO BRL). Forty-eight hours aftertransfection, the cells are split 1:1000 and 1: 5000 and if the DNAconstruct contains a neomyocin resistance gene for selection, G418 isadded to a final concentration of 0.35 mg/mL. Neomyocin resistant clonesare isolated and expanded for cyropreservation as well as nucleartransfer.

It is often desirable to express a protein, for example, a heterologousprotein, in a specific tissue or fluid, for example, the milk of atransgenic animal. A heterologous protein is one that from a differentspecies than the species being cloned. The heterologous protein can berecovered from the tissue or fluid in which it is expressed. Forexample, it is often desirable to express the heterologous protein inmilk. Methods for producing a heterologous protein under the control ofa milk-specific promoter is described below. In addition, othertissue-specific promoters, as well as, other regulatory elements, forexample, signal sequences and sequences which enhance secretion ofnon-secreted proteins, are described below. The transgenic product(e.g., a heterologous protein) can be expressed, and therefore,recovered in various tissue, cells or bodily secretions of thetransgenic animals. Examples of such tissue, cells or secretions areblood, urine, hair, skin, mammary gland, muscle, or viscera (or a tissuecomponent thereof) including, but not limited to, brain, heart, lung,kidney, pancreas, gall bladder, liver, stomach, eye, colon, smallintestine, bladder, uterus and testes. Recovery of a transgenic productfrom these tissues are well known to those skilled in the art, see, forexample, Ausubel, F. M., et al., (eds), Current Protocols in MolecularBiology, vol. 2, ch. 10 (1991).

Useful transcriptional promoters are those promoters that arepreferentially activated in mammary epithelial cells, includingpromoters that control the genes encoding protein such as caseins,β-lactoglobulin (Clark et al., Bio/Technology, 7: 487 492 (1989)), wheyacid protein (Gordon et al., Bio/Technology, 5: 1183-1187 (1987)), andlactalbumin (Soulier et al., Febs Letts., 297: 13 (1992)). Caseinpromoters may be derived from the alpha, beta, gamma, or kappa caseingenes of any animal species; a preferred promoter is derived from thegoat β-casein gene (Ditullio, Bio/Technology, 10: 74-77 (1992)). Milkspecific protein promoter or the promoters that are specificallyactivated in mammary tissue can be derived from cDNA or genomicsequences.

DNA sequence information is available for the mammary gland's specificgenes listed above, in at least one, and often in several organisms.See, for example, Richards et al., J. Biol. Chem., 256: 526-532 (1981)(α-Lactalbumin rat); Campbel et al., Nucleic Acids Res., 12: 8685-8697(1984) (rat WAP); Jones et al., J. Biol. Chem., 260: 7042-7050 (1985)(rat β-Casein); Yu-Lee and Rosen, J. Biol. Chem., 258: 10794-10804(1983) (rat α-Casein); Hall, Bio. Chem. J., 242: 735-742 (1987);(α-Lactalbumin human); Stewart, Nucleic Acids Res., 12: 389 (1984)(Bovine α S1 and θ1 Casein, cDNAs); Gorodetsky et al., Gene, 66: 87-96(1988) (Bovine β-Casein); Alexander et al., Eur. J. Biochem., 178:395-401 (1988) (Bovine and κ-Casein); Brignon et al., Febs Let., 188:48-55 (1977) (Bovine α S2 Casein); Gamieson et al., Gene, 61: 85-90(1987); Ivanov et al., Biol. Chem. Hopp-Seylar, 369: 425-429 (1988);Alexander et al., Nucleic Acid Res., 17: 6739 (1989) (Bovineβ-Lactoglobulin); Vilotte et al., Biochimie, 69: 609-620 (1987) (Bovineα-Lactalbumin).

The structure and function of the various milk protein genes arereviewed by Mercier & Vilotte, J. Dairy Sci., 76: 3079-3098 (1993). Ifadditional flanking sequences are useful in optimizing expression of theheterologous protein, such sequences can be cloned using the existingsequences as probes. Mammary gland specific regulatory sequences fromdifferent organisms can be obtained by screening libraries from suchorganisms using known cognate nucleotide sequences, or antibodies tocognate proteins as probes.

Useful signal sequences such as milk specific signal sequences or othersignal sequences which result in the secretion of eukaryotic orprokaryotic proteins can be used. Preferably, the signal sequence isselected from milk specific signal sequences, that is, it is from a genewhich encodes a product secreted into milk. Most preferably, the milkspecific signal sequence is related to the milk specific promoter usedin the construct. The size of the signal sequence is not critical. Allthat is required is that the sequence be of a sufficient size to effectsecretion of the desired recombinant protein, for example, in themammary tissue. For example, signal sequences from genes coding forcaseins, for example, α, β, γ or κ caseins and the like can be used. Apreferred signal sequence is the goat β-casein signal sequence. Signalsequences from other secreted proteins, for example, proteins secretedby kidney cells, pancreatic cells, or liver cells, can also be used.Preferably, the signal sequence results in the secretion of proteinsinto, for example, urine or blood.

A non-secreted protein can also be modified in such a manner that it issecreted such as by inclusion in the protein to be secreted all or partof the coding sequence of a protein which is normally secreted.Preferably, the entire sequence of the protein which is normallysecreted is not included in the sequence of the protein but rather onlya sufficient portion of the amino terminal end of the protein which isnormally secreted to result in secretion of the protein. For example, aportion which is not normally secreted is fused (usually at its aminoterminal end) to an amino terminal portion of the protein which isnormally secreted.

In one aspect, the protein which is normally secreted is a protein whichis normally secreted in milk. Such proteins include proteins secreted bymammary epithelial cells, milk proteins such as caseins,β-lactoglobulin, whey acid protein, and lactalbumin. Casein proteinsincluding, alpha, beta, gamma or kappa casein genes of any mammalianspecies. The preferred protein is β-casein, for example, goat β-casein.Sequences which encode the secreted protein can be derived from eithercDNA or genomic sequences. Preferably, they are of genomic origin, andinclude one or more introns.

Other tissue specific promoters which provide expression in a particulartissue can be used. Tissue specific promoters are promoters which areexpressed more strongly in a particular tissue than in others. Tissuespecific promoters are often expressed exclusively in the specifictissue.

Tissue specific promoters which can be used include: a neural-specificpromoter, for example, nestin, Wnt-1, Pax-1, Engrailed-1, Engrailed-2,Sonic-hedgehog: a liver specific promoter, for example, albumin,alpha-1, antitrypsin; a muscle-specific promoter, for example, myogenin,actin, MyoD, myosin; an oocyte specific promoter, for example, ZP1, ZP2,ZP3; a testus specific promoter, for example, protamine, fertilin,synaptonemal complex protein-1; a blood specific promoter, for example,globulin, GATA-1, porphobilinogen deaminase; a lung specific promoter,for example, surfactin protein C; a skin or wool specific promoter, forexample, keratin, elastin; endothelium-specific promoter, for example,TIE-1, TIE-2; and a bone specific promoter, for example, BMP. Inaddition, general promoters can be used for expression in severaltissues. Examples of general promoters, include β-actin, ROSA-21, PGK,FOS, c-myc, Jun-A, and Jun-B.

A cassette which encodes a heterologous protein can be assembled as aconstruct which includes a promoter for a specific tissue, for example,for mammary epithelial cells, a casein promoter. The construct can alsoinclude a 3′ untranslated region downstream of the DNA sequence codingfor the non-secreted proteins. Such regions can stabilize the RNAtranscript of the expression system and thus increase the yield ofdesired protein from the expression system. Among the 3′ untranslatedregions useful in the constructs for use in the invention are sequencesthat provide a polyA signal. Such sequences may be derived, for example,from the SV40 small t antigen, the casein 3′ untranslated region orother 3′ untranslated sequences well known in the art. In one aspect,the 3′ untranslated region is derived from a milk specific protein. Thelength of the 3′ untranslated region is not critical but the stabilizingeffect of its polyA transcript appears imported in stabilizing the RNAof the expression sequence.

Optionally, the construct can include a 5′ untranslated region betweenthe promoter and the DNA sequence encoding the signal sequence. Suchuntranslated regions can be from the same control region as that fromwhich the promoter is taken or can be from a different gene, forexample, they may be derived from other synthetic, semisynthetic ornatural sources. Again, there specific length is not critical, however,they appear to be useful in improving the level of expression.

The construct can also include about 10%, 20%, 30% or more of theN-terminal coding region of a gene preferentially expressed in mammaryepithelial cells. For example, the N-terminal coding region cancorrespond to the promoter used, for example, a goat β-casein N-terminalcoding region.

The construct can be prepared using methods known to those skilled inthe art. The construct can be prepared as part of a larger plasmid. Suchpreparation allows the cloning and selection of the correctconstructions in an efficient manner. The construct can be locatedbetween convenient restrictions sites on the plasmid so that they can beeasily isolated from the remaining plasmid sequences for incorporationinto the desired animal.

Transgenic sequences encoding heterologous proteins can be introducedinto the germ line of an animal or can be transfected into a cell lineto provide a source of genetically engineered donor cells as describedabove. The protein can be a complex or multimeric protein, for example,a homo- or hetromultimeric proteins. The protein can be a protein whichis processed by removing the N-terminus, C-terminus or internalfragments. Even complex proteins can be expressed in active form.Protein encoding sequences which can be introduced into the genome of ananimal, for example, goats, include glycoproteins, neuropeptides,immunoglobulins, enzymes, peptides and hormones. The protein may be anaturally occurring protein or a recombinant protein for example, afragment or fusion protein, (e.g., an immunoglobulin fusion protein or amutien). The protein encoding nucleotide sequence can be human ornon-human in origin. The heterologous protein may be a potentialtherapeutic or pharmaceutical agent such as, but not limited to, alpha-1proteinase inhibitor, alpha-1 antitrypsin, alkaline phosphatase,angiogenin, antithrombin III, any of the blood clotting factorsincluding Factor VIII, Factor IX, and Factor X chitinase,erythropoietin, extracellular superoxide dismutase, fibrinogen,glucocerebrosidas, glutamate decarboxylase, human growth factor, humanserum albumin, immunoglobulin, insulin, myelin basic protein,proinsulin, prolactin, soluble CD 4 or a component or complex thereof,lactoferrin, lactoglobulin, lysozyme, lactalbumin, tissue plasminogenactivator or a variant thereof. Immunoglobulin particularly preferredprotein. Examples of immunoglobulins include IgA, IgG, IgE, IgM,chimeric antibodies, humanized antibodies, recombinant antibodies,single chain antibodies and anti-body protein fusions.

Nucleotide sequence information is available for several of the genesencoding the heterologous proteins listed above, in at least one, andoften in several organisms. See, for example, Long et al., Biochem.,23(21): 4828-4837 (1984) (Alpha-1 antitrypsin); Mitchell et al., Prot.Natl. Acad. Sci. USA, 83: 7182-7186 (1986) (Alkaline phosphatase);Schneider et al., Embo J, 7(13): 4151-4156 (1988) (Angiogenin); Bock etal., Biochem., 27 (16): 6171-6178 (1988) (Antithrombin); Olds et al.,Br. J. Haematol., 78(3): 408-413 (1991) (Antithrombin III); Lyn et al.,Proc. Natl. Acad. Sci. USA, 82(22): 7580-7584 (1985) (erythropoietin);U.S. Pat. No. 5,614,184 (erythropoietin) Horowtiz, et al., Genomics,4(1): 87-96 (1989) (Glucocerebrosidase); Kelly et al., Ann. Hum. Genet.,56(3): 255-265 (1992) (Glutamate decarboxylase); U.S. Pat. No. 5,707,828(human serum albumin); U.S. Pat. No. 5,652,352 (human serum albumin);Lawn et al., Nucleic Acid Res., 9(22): 6103-6114 (1981) (human serumalbumin); Kamholz et al., Prot. Natl. Acad. Sci. USA, 83(13): 4962-4966(1986) (myelin basic protein); Hiraoka et al., Mol. Cell Endocrinol.,75(1): 71-80 (1991) (prolactin); U.S. Pat. No. 5,571,896 (lactoferrin);Pennica et al., Nature, 301(5897): 214-221 (1983) (tissue plasminogenactivator); Sarafanov et al., Mol. Biol., 29: 161-165 (1995).

A transgenic protein can be produced in the transgenic cloned animal atrelatively high concentrations and in large volumes, for example inmilk, providing continuous high level output of normally processedprotein that is easily harvested from a renewable resource. There areseveral different methods known in the art for isolation of proteins formilk.

Milk proteins usually are isolated by a combination of processes. Rawmilk first is fractionated to remove fats, for example by skimming,centrifugation, sedimentation, (H. E. Swaisgood, Development in DairyChemistry, I: Chemistry of Milk Protein, Applied Science Publishers, NY1982), acid precipitation (U.S. Pat. No. 4,644,056) or enzymaticcoagulation with rennin or chymotrypsin (Swaisgood, ibid.). Next themajor milk proteins may be fractionated into either a clear solution ora bulk precipitate from which this specific protein of interest may bereadily purified.

French Patent No. 2487642 describes the isolation of milk proteins fromskim milk or whey by performing ultra filtration in combination withexclusion chromatography or ion exchange chromatography. Whey is firstproduced by removing the casein by coagulation with rennet or lacticacid. U.S. Pat. No. 4,485,040 describes the isolation of anα-lactoglobulin-enriched product in the retentate from whey by twosequential ultra filtration steps. U.S. Pat. No. 4,644,056 provides amethod for purifying immunoglobulin from milk or colostrum by acidprecipitation at pH 4.0-5.5, is sequential cross-flow filtration firston a membrane with 0.1-1.2 mm pore size to clarify the product pool andthen on a membrane with a separation limit of 5-80 kD to concentrate it.Similarly, U.S. Pat. No. 4,897,465 teaches the concentration andenrichment of a protein such as immunoglobulin from blood serum, eggyolks or whey by sequential ultra filtration on metallic oxide membraneswith a pH shift. Filtration is carried out first at a pH below theisoelectric point (pI) of the selected protein to remove bulkcontaminants from the protein retentate, in next adding pH above the pIof the selected protein to retain impurities and pass the selectedprotein to the permeate. A different filtration concentration method istaught by European Patent No. EP 467 482 B 1 in which defatted skim milkis reduced to pH 3-4, below the pI of the milk proteins, to solubilizeboth casein and whey proteins. Three successive rounds of ultrafiltration are diafiltration and concentrate the proteins to form aretentate containing 15-20% solids of which 90% is protein.Alternatively, British Patent Application No. 2179947 discloses theisolation of lactoferrin from whey by ultra filtration to concentratethe sample, fall by week cation exchange chromatography at approximatelya neutral pH. No measure of purity is reported in PC Publication No. WO95/22258, a protein such as lactoferrin is recovered from milk that hasbeen adjusted to high ionic strength by the addition of concentratedsalt, followed by cation exchange chromatography.

In all these methods, milk or a fraction thereof is first treated toremove fats, lipids, and other particular matter that would foulfiltration membranes or chromatography medium. The initial fractionsthus produce can consist of casein, whey, or total milk protein, fromwhich the protein of interest is then isolated.

PCT Patent Publication No. WO 94/19935 discloses a method of isolating abiologically active protein from whole milk by stabilizing thesolubility of total milk proteins with a positively charged agent suchas arginine, imidazole or Bis-Tris. This treatment forms a clarifiedsolution from which the protein may be isolated for example byfiltration through membranes that otherwise would become clogged byprecipitated proteins.

Methods for isolating a soluble milk component such as a peptide in itsbiologically active form from whole milk or a milk fraction bytangential flow filtration are known. Unlike previous isolation methods,this eliminates the need for a first fractionation of whole milk toremove fat micelles, thereby simplifying the process in avoiding lossesof recovery of bioactivity. This method may be used in combination withadditional purification steps to further remove contaminants and purifythe product (e.g., the protein of interest).

Another aspect of the present invention includes methods for enucleatingan activated oocyte comprising contacting the oocyte with a compoundthat destabilizes (e.g., disrupts or disassociates) the meiotic spindleapparatus. Disruption of the meiotic spindle apparatus results indisruption of microtubules, chromosomes and centrioles. Such a compoundrenders the nucleus non-functional. Examples of such compounds arecochicine, pactiltaxel, nocodazole and preferably, demecolcine.

This aspect of the invention can be used for enucleation in combinationwith the methods described herein. For example, an activated oocyte canbe prepared for nuclear transfer by activating the oocyte (e.g.,exposing the oocyte to ethanol or an ionophore), and then subjecting theactivated oocyte to a compound that destabilizes the meiotic spindles(e.g., demecolcine). Once the activated oocyte is prepared, then it canbe combined with genome from an activated donor cell to result in anuclear transfer embryo.

The following examples are intended to be illustrative and not limitingin any way.

Exemplification

EXAMPLE 1 Cloning Mice Using an Induced Enucleation Method

Advances in somatic cell nuclear transfer (NT) methodology have seen theprocession of cloned sheep, cows, mice and goats. Despite the clearpotential use of this technology for genetic manipulations, the successrates remain woefully low. The limitations associated with nucleartransfer include the selection and preparation of competent donorcytoplasts. The cytoplasm plays a vital role in genome reprogramming andreactivation and therefore manipulations that compromise oocytedevelopmental competence are detrimental to the success of nucleartransfer. The present study was directed towards determining alternativemethods to more efficiently prepare competent cytoplasts for nucleartransfer procedures. In experiment 1, in vivo-produced mouse metaphaseII oocytes (B6D2, aguti, recovered at 16-20 h post hCG) were activatedby exposure to either 7% ethanol or 2 μm ionomycin in PBS+10% FBS (5min.). At the onset of initial second polar body formation (10-15 min.post activation) and extrusion (anaphase/telophase stage), oocytes wererandomly allocated to control (cultured 0.5-1.5 h) or incubated withTaxol® (5 μg/ml), Cycloheximide (10 μg/ml) or in Demecolcine (0.4 μg/ml)in PBS+10% FBS until the second polar body was extruded (0.5-1.5 h postactivation) to induce nuclear chromatin enucleation. Oocytes werestained (H33342, 5 μg/ml) to confirm the extent of nuclear chromatinenucleation using fluorescence microscopy. The rate of treatment-inducedchromatin enucleation was 3.7% (0-15%) for control, 3.6% (0-10%) forTaxol®, 16.3% (0-24%) for Cycloheximide and 54% (27-70%) for Demecolcinetreatments. In experiment 2, Demecolcine-induced enucleated cytoplastswere used for nuclear transfer recipients. Donor nuclei were preparedfrom cumulus cells (Black Swiss) by partial lysis (1% sodium citrate)followed by aspiration using the injection pipette (7 um). Donor nucleiwere injected into cytoplasts pretreated with Cytochalasin-B (5 μg/ml,15 min.). Reconstructed NT embryos were subsequently co-cultured (72-96h) with oviductal cells in drops of M-199+10% FBS. The cleavage rate was70% (85/121) and the rate of blastocyst formation was 42% (51/121).Pregnancies were established in 2/3 recipients (CD1, white) followinguterine embryo transfer (10 embryos/recipient). A total of 14 blackfemale pups were born (47%, 14/30), seven of which were stillborn from 1recipient and the other seven were born live (23%, 7/30) but 3 werecannibalized within 24 h. The 4 cloned pups were normal and healthy, andtheir fertility is being assessed. These data suggest that cytoskeletonmodifying agents can induce enucleation of nuclear chromatin atacceptable rates without physical perturbation associated withmechanical enucleation and with no loss of cytoplasm. Moreover,cytoplasts derived from this enucleation procedure are competent tosupport genome reactivation and fetal development to term. Thistechnically simple approach may provide a more efficient method toproduce large numbers of cytoplasts for cloning procedures.

EXAMPLE 2 Cloning a Transgenic Goat

Donors and recipients used in the following examples were dairy goats ofthe following breeds (mixed or not): Alpine, Saanen, and Toggenburg.Collections and transfers were completed during the spring and earlysummer (off-season).

Isolation of Caprine Somatic Cells

Caprine fetal fibroblast cell lines used as karyoplast donors werederived from six day 35-40 fetuses produced by artificially inseminatingnon-transgenic does with fresh collected semen from a transgenicantithrombin III (ATIII) founder buck. An ATIII cell line was chosensince it provides a well characterized genetic marker to the somaticcell lines, and it targets high level expression of a complexglycosylated protein (ATIII) in the milk of lactating does. Threefetuses which were derived from the semen of the transgenic ATIII buckwere surgically removed at day 40 post coitus and placed in equilibratedCa⁺⁺/Mg⁺⁺-free phosphate buffered saline (PBS). Cell suspensions wereprepared by mincing and digesting fetal tissue in 0.025% trypsin/0.5 mMEDTA at 37° C. for ten minutes. Cells were washed with equilibratedMedium 199™ (M199)(Gibco)+10% Fetal Bovine Serum (FBS) supplemented withnucleosides, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1%penicillin/streptomycin (10,000 U.U. each/ml) (fetal cell medium), andcultured in 25 cm² flasks. The cultures were re-fed 24 hours later withequilibrated fetal cell medium. A confluent monolayer of primary fetalcells was harvested by trypsinization on day four by washing themonolayer twice with Ca⁺⁺/Mg⁺⁺-free PBS, followed by incubation with0.025% trypsin/0.5 mM EDTA at 38° C. for 7 minutes.

Cells potentially expressing ATIII were then prepared forcryopreservation, or maintained as stock cultures.

Sexing and Genotyping of Donor Cell Lines

Genomic DNA was isolated from fetal head tissue for ATIII donorkaryoplasts by digestion with proteinase K followed by precipitationwith isopropanol as described in Laird et al. (1991) Nucleic Acid Res.19: 4293, and analyzed by polymerase chain reaction (PCR) for thepresence of human Antithrombin III (ATIII) sequences. The ATIII sequenceis part of the BC6 construct (Goat Beta-casein-humanATIII cDNA) used togenerate the ATIII transgenic line as described in Edmunds et al. (1998)Blood 91: 4561-4571. The human ATIII sequence was detected byamplification of a 367 bp sequence with oligonucleotides GTC11 and GTC12(see below). For sexing, the zfX/zfY primer pair was used (see below)giving rise to a 445 pb (zfX)/447 bp (sfy) doublet. Upon digestion withthe restriction enzyme SacI (New England Biolabs), the zfX band was cutinto two small fragments (272 and 173 bp). Males were identified by thepresence of the uncut 447 bp zfY band.

For the PCR reactions, approximately 250 ng of genomic DNA was dilutedin 50 ml of PCR buffer (20 mM Tris pH 8.3, 50 mM KCl and 1.5 mM MgCl₂,0.25 mM deoxynucleotide triphosphates, and each primer at aconcentration of 600 mM with 2.5 units of Taq polymerase and processedusing the following temperature program.  1 cycle at 94° C. 60 seconds 5 cycles at 94° C. 30 seconds 58° C. 45 seconds 74° C. 45 seconds 30cycles at 94° C. 30 seconds 55° C. 30 seconds 74° C. 30 seconds

The following primer set was used to detect the human ATIII sequence:GTC 11: CTCCATCAGTTGCTGGAGGGTGTCATTA (SEQ ID NO: 1) GTC 12:GAAGGTTTATCTTTTGTCCTTGCTGCTCA (SEQ ID NO: 2)

The following primer set was used for sexing: zfX:ATAATCACATGGAGAGCCACAAGC (SEQ ID NO: 3) zfY: GCACTTCTTTGGTATCTGAGAAAG(SEQ ID NO: 4)

Two of the fetuses were identified to be male and were both negative forthe ATIII sequence. Another fetus was identified as female and confirmedpositive for the presence of the ATIII sequence.

Preparation of ATIII-Expressing Donor Cells for Embryo Reconstitution

A transgenic female line (CFF155-92-6) originating from a day 40 fetuswas identified by PCR analyses, as described above, and used for allnuclear transfer manipulations. Transgenic fetal fibroblast cells weremaintained in 25 cm² flasks with fetal cell medium, re-fed on day fourfollowing each passage, and harvested by trypsinization on day seven.From each passage, a new 25 cm² flasks was seeded to maintain the stockculture. Briefly, fetal cells were seeded in 4-well plates with fetalcell medium and maintained in culture (5% CO₂ at 39° C.), forty-eighthours later, the medium was replaced with fresh fetal cell mediumcontaining 0.5% FBS. The culture was re-fed every 48-72 hours over thenext seven days with fresh fetal cell medium containing 0.5% FBS. On theseventh day following first addition of fetal cell medium (0.5% FBS),somatic cells used as karyoplast donors were harvested by trypsinizationas previously described. The cells were resuspended in equilibratedM199+10% FBS supplemented with 2 mM L-glutamine, 1%penicillin/streptomycin (10,000 I.U. each/ml) one to three hours priorto fusion to the enucleated oocytes.

Karyotyping of Cell Lines

The clonal lines were further evaluated by karyotyping to determinegross chromosomal abnormalities in the cell lines. Cells were induced toarrest at metaphase by incubation with 0.02 μg/ml of Demecolcine (Sigma)for 12 hours. After trypsinization, the resulting pellet was suspendedin a hypotonic solution of 75 mM KCl in water and incubated at 37° C.for 20 minutes. Cells were fixed for 5 minutes each time in 3 changes ofice-cold acetic acid-methanol (1:3) solution before drops of the cellsuspension were place din pre-washed microscopic slides. Followingair-drying, chromosome preparations were stained with 3% Giemsa stain(Sigma) in PBS for 10 minutes. The chromosome spreads were counted foreach cell line at 1000× magnification under oil immersion.

Immunohistochemical Analysis

Antibodies specific for vimentin (Sigma) and pan-cytokeratin (Sigma)were used to characterize and confirm the morphology of the cell lines.Cells were plated in sterile gelatin coated cover slips to 75%confluence and fixed in 2% paraformaldehyde with 0.05% saponin for 1hour. Cells were incubated in 0.5% PVP in PBS (PBS/PVP) with primaryantibodies for 2 hours at 37° C., rinsed with 3 changes of PBS/PVP at 10minute intervals, and incubated for 1 hour in secondary antibodiesconjugated with Cy3 and FITC respectively. Alkaline phosphatase (Sigma)activity of the cells was also performed to determine the presence orabsence of undifferentiated cells. The coverslips were rinsed andsubsequently mounted on glass slides with 50% glycerol in PBS/PBP with10 μg/ml bisbenzimide (H-33342, Sigma) and observed under fluorescentmicroscopy.

Epithelial and fibroblast lines positive for fimentin andpan-cytokeratin, respectively, and negative for alkaline phosphataseactivity were generated from the ATIII primary cultures. In the cellcultures, two morphologically distinct cell types were observed. Larger“fibroblast-like” cells stained positive for vimentin and smaller“epithelial-like” cells stained positive for pan-cytokeratin whichcoexisted in the primary cell cultures. The isolated fibroblast linesfrom ATIII showed a tendency to differentiate into epithelial-like cellswhen cultured for 3 days after reaching confluency. Subsequent passagesinduced selection against fibroblast cells giving rise to pureepithelial cells as confirmed by the lack of positive staining forvimentin. Senesces or possible cell cycle arrest was first observed atpassage 28. These cells appear bigger in size (>30 μm) compared to thenormally growing cells (15-25 μm) and can be maintained in culture inthe absence of apparent mitotic activity for several months without lossof viability. Embryo reconstruction using nuclei from the arrested cellsproduced morula stage embryos suggesting reacquisition of mitoticactivity.

Donor Karyoplast Cell Cycle Synchronization and Characterization

Selected diploid transgenic female cell lines were propagated, passagedsequentially and cyrobanked as future karyoplast stock. Donor karoplastsfor nuclear transfer were seeded in 4 well plates and cultured for up to48 hours in DMEM+10% FBS or when cells reached 70-80% confluency.Subsequently, the cells were induced to exit growth phase and enter thequiescent stage (G₀) by serum deprivation for seven days using DMEMsupplemented with 0.5% FBS to synchronize the cells. Followingsynchronization at G₀, a group of cells were induced to re-enter thecell cycle by resuspending the cells in M199+10% FBS up to three hoursprior to karyoplast-cytoplast fusion to synchronize the cells at theearly G₁ phase prior to START. A second group of cells were alsoreleased from the quiescent state and cultured in M199+10% FBS for 12 or36 hours to synchronize cells at the S-phase. Cells were harvested bystandard trypsinization and resuspended in M199+10% FBS and electrofusedas karyoplasts donors within 1 hour.

The metaphase spreads from the transgenic cell lines carrying the ATIIIconstruct at passage 5 was 81% diploid and this did not altersignificantly at passage 15 where 78% of the spreads were diploid.

Cell cycle synchrony was determined by immunohistochemical analysisusing antibodies against cyclin D1, 2, 3 and PCNA (Oncogene ResearchProducts) for the absence of protein complex to indicate quiescence (G₀)or presence of the complex to indicate G₁ entry. Cells in the presumedS-phase of the cell cycle were identified by the presence of DNAsynthetic activity using the thymidine analog 5-bromo2′-deoxyuridine-5′triphospate (BrDu, Sigma) and streptavidin-Biotin BrDustaining kit (Oncogene Research Products).

Immunofluorescence analysis of cells subjected to the synchronizationregimen demonstrated that following seven days of serum deprivation, 90%of the cells were negative for G₁ stage cyclins D1, 2, 3 and PNCA, andwere therefore in G₀ arrest. Restoration of the serum content to 10% forthis line induced reentry into the cell cycle with approximately 74% ofthe cells reaching early G₁ within 3 hours following serum additionbased on positive staining for cyclin D1. Serum restoration for 12 and36 hours showed that 89% of the cells were positive for BrDu indicatingDNA synthetic activity. In this study, clonal lines generally respondeddifferently to the serum synchronization regimen. An indirectrelationship was observed where the rate of cell synchronizationdecreases with the increase in passage numbers. Further, as passagenumber increased the population doubling times decreased, each clonalcell line revealed a decreased sensitivity to serum synchronization ofthe cell cycle.

Superovulation of Donor Goats and Oocyte Collection

Estrus was synchronized on day 0 by a 6 mg subcutaneous Norgestomet earimplant (Synchro-mate B). A single injection of prostaglandinn (PGF2α)(Upjohn US) was administered on day 7. Starting on day 12, FSH(Folltropin-V, Vetrepharm, St. Laurent, Quebec, Canada) was administeredtwice daily over four consecutive days. The ear implant was removed onday 14. Twenty-four hours following implant removal, the donor animalswere mated several times to vasectomized males over a 48 hour interval.A single injection of GnRH (Rhone-Merieux US) was administeredintramuscularly following the last FSH injection. Oocytes were recoveredsurgically from donor animals by mid-ventral laparotomy approximately 18to 24 hours following the last mating, by flushing the oviduct withCa⁺⁺/Mg⁺⁺-free PBS prewarmed at 37° C. Oocytes were then recovered andcultured in equilibrated M199+10% FBS supplemented with 2 mML-glutamine, 1% penicillin/streptomycin (10,000 I.U. each/ml).

Oocyte Enucleation

In vivo matured oocytes were collected from donor goats. Oocytes withattached cumulus cells or devoid of polar bodies were discarded.Cumulus-free oocytes were divided into two groups: oocytes with only onepolar body evident (metaphase II stage) and the activated telophase IIprotocol (oocytes with one polar body and evidence of an extrudingsecond polar body). Oocytes in telophase II were cultured in M199+10%FBS for 3 to 4 hours. Oocytes that had activated during this period, asevidenced by a first polar body and a partially extruded second polarbody, were grouped as culture induced, calcium activated telophase IIoocytes (Telophase II-Ca⁺²) and enucleated. Oocytes that had notactivated were incubated for 5 minutes in PBS containing 7% ethanolprior to enucleation. Metaphase II stage oocytes (one polar body) wereenucleated with a 25-30 micron glass pipette by aspirating the firstpolar body and adjacent cytoplasm surrounding the polar body(approximately 30% of the cytoplasm) presumably containing metaphaseplate.

As discussed above, telophase stage oocytes were prepared by twoprocedures. Oocytes were initially incubated in phosphate bufferedsaline (PBS, Ca⁺²/Mg⁺² free) supplemented with 5% FBS for 15 minutes andcultured in M199+10% FBS at 38° C. for approximately three hours untilthe telophase spindle configuration or the extrusion of the second polarbody was reached. All the oocytes that responded to the sequentialculture under differential extracellular calcium concentration treatmentwere separated and grouped as Telophase II-Ca²⁺. The other oocytes thatdid not respond were further incubated in 7% ethanol in M199+10% FBS for5-7 minutes (Telophase II-ETOH) and cultured in M199+10% FBS for 2 to 4hours. Oocytes were then cultured in M199+10% FBS containing 5 μg/ml ofcytochalasin-B for 10-15 minutes at 38° C. Oocytes were enucleated witha 30 micron (OD) glass pipette by aspirating the first polar body andapproximately 30% of the adjacent cytoplasm containing the metaphase IIor about 10% of the cytoplasm containing the telophase II spindle. Afterenucleation the oocytes were immediately reconstructed.

Embryo Reconstruction

CFF 155-92-6 somatic cells used as karyoplast donors were harvested onday 7 by trypsinizing (0.025% trypsin/0.5 mM EDTA) (Sigma) for 7minutes. Single cells were resuspended in equilibrated M199+10% FBSsupplemented with 2 mM L-glutamine, penicillin/streptomycin. The donorcell injection was carried out inn the same medium as for enucleation.Donor cells were graded into small, medium and large before selectionfor injection to enucleated cytoplasts. Small single cells (10-15micron) were selected with a 20-30 micron diameter glass pipette. Thepipette was introduced through the same slit of the zona made duringenucleation and donor cells were injected between the zone pellucida andthe ooplasmic membrane. The reconstructed embryos were incubated in M19930-60 minutes before fusion and activation.

Fusion and Activation

All reconstructed embryos (ethanol pretreatment or not) were washed infusion buffer (0.3 M mannitol, 0.05 mM CaCl₂, 0.1 mM MgSO₄, 9 mM K₂HPO⁴,0.1 mM glutathione, 0.1 mg/ml BSA in distilled water) for 3 minutesbefore electrofusion. Fusion and activation were carried out at roomtemperature, in a chamber with two stainless steel electrodes 200microns apart (BTX® 200 Embryomanipulation System, BTX®-Genetronics, SanDiego, Calif.) filled with fusion buffer. Reconstructed embryos wereplaced with a pipette in groups of 3-4 and manually aligned so thecytoplasmic membrane of the recipient oocytes and donor CFF155-92-6cells were parallel to the electrodes. Cell fusion and activation weresimultaneously induced 32-42 hours post GnRH injection with an initialalignment/holding pulse of 5-10 V AC for 7 seconds, followed by a fusionpulse of 1.4 to 1.8 KV/cm DC for 70 microseconds using an ElectrocellManipulator and Enhancer 400 (BTX®-Genetronics). Embryos were washed infusion medium for 3 minutes, then they were transferred to M199containing 5 μg/ml cytochalasin-B (Sigma) and 10% FBS and incubated for1 hour. Embryos were removed from M199/cytochalasin-B medium andco-cultured in 50 microliter drops of M199 plus 10% FBS with goatoviductal epithelial cells overlaid with paraffin oil. Embryo cultureswere maintained in a humidified 39° C. incubator with 5% CO₂ for 48hours before transfer of the embryos to recipient does.

Reconstructed embryos at 1 hour following simultaneous activation andfusion with G₀, G₁ and S-phase karyoplasts all showed nuclear envelopebreakdown (NEBD) and premature chromosome condensation (PCC) when thecytoplasts were at the arrested metaphase II stage. Subsequent nuclearenvelope formation was observed to be at about 35% at 4 hour postactivation. Oocytes reconstructed at telophase II stage showed that anaverage of 22% of oocytes observed at 1 hour post fusion of G₀, G₁ andS-phase karyoplast underwent NEBD and PCC, whereas the remaining oocyteshave intact nuclear lamina surrounding the decondensing nucleus. Noconsistent nuclear morphology other than lack of, or the occurrence ofNEBD and PCC was observed between the metaphase and two telophasereconstruction protocols employed. Differences became evident whencloned embryos were observed to have a higher incidence of advancedcleavage stages (8 to 32 blastomeres) when embryos were reconstructedwith S-phase donor nuclei compared to when G₀ or G₁ stage karyoplastswere used (2 to 8 blastomeres) following culture in vitro for 36 to 48hours. Fluorescent microscopy analysis showed that the nuclei of some ofthe rapidly dividing embryos were fragmented. Other embryos developed tothe 32 to 64 cell stage within 3 days of culture before cleavagedevelopment was blocked. Analysis of blastomere and nuclei numbers ofthese embryos showed the failure of synchronous occurrence of cytokinesand karyokinesis wherein blastomeres were either devoid or theircorresponding nuclei or contained multiple nuclei. In contrast,morphologically normal looking embryos showed synchronous cytokinesisand karyokinesis.

Goat Oviductal Epithelial Cells (GOEC) Reconstructed Embryo Co-Culture

GOEC were derived from oviductal tissue collected during surgicaloviductal flushing performed on synchronized and superovulated does.Oviductal tissue from a single doe was transferred to a sterile 15 mlpolypropylene culture tube containing 5 ml of equilibrated M199, 10%FBS, 2 mM L-glutamine, penicillin/strepomycin. A single cell suspensionwas prepared by vortex mixing for 1 minute, followed by culture in ahumidified 5% CO₂ incubator at 38° C. for up to one hour. The tube wasvortex mixed a second time for one minute, then cultured an additionalfive minutes to allow debris to settle. The top four millimeterscontaining presumed single cells was transferred to a new 15 ml culturetube and centrifuge at 600×g for 7 minutes, at room temperature. Thesupernatant was removed, and the cell pellet resuspended in 8 ml ofequilibrated GOEC medium. The GOEC were cultured in a 25 cm² flask,re-fed on day 3, and harvested by trypsinization on day six, aspreviously described. Monolayers were prepared weekly, from primary GOECcultures, for each experiment. Cells were resuspended in GOEC medium at5×10⁵/ml, and 50 microliter/well was seeded in 4-23 ll plates (15 mm).The medium was overlaid with 0.5 ml light paraffin oil, and the plateswere cultured in a humidified 5% CO₂ incubator at 38° C. The cultureswere re-fed on day two with 80% fresh equilibrated culture medium. Allreconstructed embryos were co-cultured with the GOEC monolayers in vitroin incubator at 39° C., 5% CO₂ before transfer to recipients at GTCfarm.

All experimental replicates for ATIII yielded cleavage stage embryosthat were transferable on day 2 into synchronized recipients. Embryosusing fibroblasts and epithelial cell phenotype as donor karyoplastsshowed cleavage and development in culture. The percentage of cleavagedevelopment was higher in reconstructed couplets that used preactivatedtelophase II stage cytoplasts (45%) and telophase II-ethanol activated(56%) when compared to cytoplasts used at metaphase II arrested (35%)using ATIII karyoplasts. There were no differences observed in thecleavage rates of embryos that were reconstructed using donorkaryoplasts in G₀, G₁ or S-phase of the cell cycle although, themorphological quality of embryos was better when donor karyoplasts werein as G₀ or G₁ compared to S-phase. Embryos were generally between the 2to 8 cell stage with the majority of the embryos having 3-4 blastomeresat the time of transfer. Normal cleavage development correspondedchronologically to approximately 36 to 48 hours post fusion andactivation. Morphologically normal appearing embryos were selected atthe 2 to 8 cell stage following development in vitro for 36 to 48 hours.

Estrus Synchronization of Recipient Does

Hormonal treatments were delayed by 1 day for recipients (as compared todonor) to insure donor/recipient synchrony. Estrus was synchronized onday 1 by a 6 mg subcutaneous norgestomet ear implant. A single injectionof prostaglandin was administered on day 8. Starting on day 14, a singleintramuscular treatment of PMSG (CalBiochm US) was administered. The earimplant was removed on day 15. Twenty-four hours following implantremoval, recipient does were mated several times to vasectomized malesover three consecutive days.

Embryo Transfer to Recipient Does

Reconstructed embryos were co-cultured with GOEC monolayers forapproximately 48 hours prior to transfer to synchronized recipients.Immediately prior to transfer, reconstructed embryos were placed innequilibrated Ham's F-12 medium+10% FBS. Two to four reconstructedembryos were transferred via the fimbria into the oviductal lumen ofeach recipient. Transfers were performed in a minimal volume of Hams'sF-12 medium+10% FBS using a sterile fire-polished glass micropipette.

The development of embryos reconstructed by nuclear transfer usingtransgenic caprine fetal fibroblasts and in vivo derived oocytes issummarized in Table 1. There was a total of 14 rounds of collection andtransfers, with 4 donors set up for collection and 5-6 recipient doesset up for transfer 48 hours later. The three differentenucleation/activation protocols were employed: Metaphase II, Telophase,and Metaphase II pretreated with Ethanol. Following fusion-activation,reconstructed embryos were co-cultured with primary goat epithelialcells, at least until cleavage (2-cell stage) up to early 16-cell stage;with most embryos being transferred at chronologically correct 2- and4-cell stages. All transfers were surgical and oviductal, in hormonallysynchronized recipients (due to the season). Rates of development wereslightly superior when using Telophase protocol and Ethanol protocol ascompared to the Metaphase II protocol. This is partly due to the factthat enucleation of the second polar body seems less traumatic for theoocytes, and partly due to what seems to be higher activation rate foroocytes pretreated with ethanol. TABLE 1 Development of caprine embryosreconstructed by nuclear transfer of transgenic fetal fibroblasts. Threeenucleation/procedure were used: Metaphase II (first polar bodyenucleation), Telophase (second polar body enucleation), Ethanol(preactivation of Metaphase II stage oocytes by 7% ethanol treatmentprior to enucleation). In all cases, concomitant fusion and activationwas used. Enucleation Embryos and Activation Oocytes Oocytes CleavedEmbryos Protocol Reconstructed Lysed (%) (%) Transferred Metaphase II138   67 (48.5) 48 (35) 47 Telophase- 92 38 (41) 41 (44) 38 Ca²⁺Telophase- 55 23 (42) 31 (56) 27 EtOH

Following embryo transfer, recipient does were examined by ultrasound,as early as day 25. High pregnancy rates ranging from 55-78% for ATIIIrecipient does were diagnosed. For all three enucleation/activationprotocols, it was observed that high proportion of does (65%) appearedpositive at day 30. However, it must be noted that, in most cases, fetalheartbeats could not be detected at such an early stage. Moreover, thepositive ultrasound signal detected at day 30 was not characteristic ofnormal embryo development and appeared closer to vesicular developmentnot associated with the formation of an embryo proper. This kind ofembryonic development is not typically observed in other caprine embryotransfer programs (for example with microinjected embryos). Biweekly,examination of these vesicular developments between day 25 and 40established that these pregnancies were abnormal and at day 40, most ofthe fetuses were reabsorbed and normal ultrasound images were notapparent.

However, for 2 pregnancies, heartbeats were detected by day 40. In these2 cases, ultrasound examination between day 25 and day 40, not onlydetected a heartbeat, but also showed the development of recognizableembryonic structures. One of these pregnancies was established using theMetaphase II enucleation/activation protocol, fusing the enucleatedcytoplast to a quiescent karyoplast originating from a passage 6 cultureof the CFF155-92-6 fibroblast cell line. In this instance, 4 four-cellstage reconstructed embryos were transferred to the oviduct of therecipient doe. The other pregnancy (twins) was obtained from embryosreconstructed according to the Telophase enucleation/activationprotocol, fusing an enucleated cytoplast derived from preactivatedtelophase Ca²⁺ oocytes and G₁ karyoplasts originating from a passage 5culture of the CFF155-92-6 epithelial cell line. In this case, 3reconstructed embryos (1 two-cell stage and 2 four-cell stage) weretransferred to the oviduct of the recipient doe.

No pregnancies were observed with embryos generated by the Ethanolenucleation/activation protocol. However, numbers are not large enoughto conclude on the relative efficacy of the 3 enucleation/activationprotocols used in this study. TABLE 2 Induction of pregnancy and furtherdevelopment following transfer of caprine embryos reconstructed withtransgenic fetal fibroblasts and activated according to three protocols.Enucleation Recipients Ultrasound Results Term Activation (average # of(positive/total recip) Preg- Protocol embryos/recip) 30 days 40 days 50days nancies Metaphase II 15 (3.1)  9/15 1/15 1/15 1 Telophase-Ca²⁺ 14(2.7) 11/14 1/14 1/14 1 (twins) Telophase-EtOH 9 (3)  5/9 0/9  0/9  0Perinatal Care of Recipient Embryos

Does were monitored daily throughout pregnancy for outward signs ofhealth (e.g., appetite, alertness, appearance). Pregnancy was determinedby ultrasonograph 25-28 days after the first day of standing estrus.Does were subjected to ultrasound biweekly till approximately day 75 andthere after once a month to monitor the assess fetal viability.Additionally, recipient does had serum samples drawn at approximated day21 post standing estrus for serum progesterone analysis. This was todetermine if a functioning corpus luteum was present and how thiscompared to the animal's reproductive status (i.e., pregnancy). Atapproximately day 130, the pregnant does were vaccinated with tetanustoxoid and Clostridium C&D. Selenium & vitamin E (Bo-Se) and vitamins A,D, and B complex were given intramuscularly or subcutaneously and ade-wormer was administered. The does were moved to a clean kidding stallon approximately day 143 and allowed to acclimate to this newenvironment prior to kidding. Observations of the pregnant does wereincreased to monitor for signs of pending parturition. After thebeginning of regular contractions, the does remained under periodicobservation until birth occurred. If labor was not progressive afterapproximately 15 minutes of strong contractions the fetal position wasassessed by vaginal palpation. If the position appeared normal then thelabor was allowed to proceed for an additional 5-30 minutes (dependingon the doe) before initiating an assisted vaginal birth. If indicated acesarean section was performed. When indicated, parturition was inducedwith approximately 5-10 mg of PGF2α (e.g. Lutalyse). This induction canoccur approximately between 145-155 days of gestation. Parturitiongenerally occurs between 30 and 40 hours after the first injection. Themonitoring process is the same as described above.

Once a kid was born, the animal was quickly towel dried and checked forgross abnormalities and normal breathing. Kids were immediately removedfrom the dam. Once the animal was determined to be in good health, theumbilicus was dipped in 7% tincture of iodine. Within the first hour ofbirth, the kids received their first feeding of heat-treated colostrum.At the time of birth, kids received injections of selenium & vitamin E(Bo-Se) and vitamins A, D, and B complex to boost performance andhealth.

The first transgenic female goat offspring produced by nuclear transferwas born after 154 days of gestation following the induction ofparturition and cesarean delivery. The birth weight of the offspring was2.35 kg which is with the medium weigh range of the alpine breed. Thefemale twins were born naturally with minimal assistance a month laterwith a gestation length of 151 days. The birth weights of the twins wereboth 3.5 kg which are also within the medium weight range for twins ofthis breed. All three kids appeared normal and healthy and werephenotypically similar for coat color and expressing markings typical ofthe alpine breed. In addition, all three offspring were similar inappearance to the transgenic founder buck. No distinguishable phenotypicinfluence from the breed of the donor oocyte (Saanen, Toggenburg breed)or the heterogeneous expression of the fetal genotype was observed.

Transgenic Cloned Goats

In order to confirm that the three kids were transgenic for the BC6construct comprising the goat beta casein promoter and the human ATIIIgene sequence, PR amplification and southern analysis of the segment ofthe transgene were performed.

Shortly after birth, blood samples and ear skin biopsies were obtainedfrom the cloned female goats and the surrogate dams. The samples weresubjected to genomic DNA isolation. Laird et al., Nucleic Acids Res.,19: 4293 (1991). Each sample was first analyzed by PCR using ATIIIspecific primers, and then subjected to Southern blot analysis using theATIII cDNA (Edmunds et al., Blood, 91: 4561-4571 (1998). For eachsample, 5 μg of genomic DNA was digested with EcoRI (New EnglandBiolabs, Beverly, Mass.), electrophoresed in 0.7% agarose gels (SeaKam®,ME) and immobilized on nylon membranes (MagnaGraph, MSI, Westboro,Mass.) by capillary transfer following standard procedures (Laird etal., Nucleic Acids Res., 19: 4293 (1991). Membranes were probed with the1.5 kb XhoI to SalI ATIII cDNA fragment labeled with α³²p dCTP using thePrime-It® kit (Stratagene, La Jolla, Calif.). Hybridization was executedat 65° C. overnight (Church et al., Prot. Natl. Acad. Sci. USA, 81:1991-1995 (1984). The blot was washed with 0.2×SSC, 0.1% SDS and exposedto X−)MAT™ AR film for 48 hours.

PCR analysis confirmed that all of the kids were transgenic for the BC6construct comprising the goat beta casein promoter and the human ATIIIgene sequence. Southern blot analysis demonstrated the integrity of theBC6 transgene. Hybridization to a diagnostic 4.1 kb EcoRI fragment wasdetected for all three cloned animals, the cell lines and a transgenicpositive control, but not for the two recipient does. As expected, dueto cross hybridization of the ATIII cDNA probe to the endogenous goat ATlocus, a 14 kb band was detected in all samples.

In addition, fluorescence in situ hybridization (FISH) was performed todetermine the integration site of the BC6 construct. For typing of thecloned goats, whole blood was cultured for lymphocytes harvest (Ponce deLeon et al., J. Hered., 83: 36-42 (1992). Fibroblast cells andlymphocytes were pretreated and hybridized as previously described invan de Corput et al., Histochem Cell Biol., 110: 431-437 (1998), andKlinger et al., Am. J. Human. Genet., 51: 55-65 (1992). A digoxygenlabeled probe containing the entire 14.7 kb BC6 transgene was used inthis procedure. The TSA™ Direct system (NEN™ Life Science Products,Boston, Mass.) was used to amplify the signal. R-bands were visualizedusing DAPI counterstain and identified as in Di Berardino et al., J.Hered., 78: 225-230 (1987). A Zeiss Axioskop microscope mounted with aHamamatsu Digital Camera was used with Image-Pro® Plus software (MediaCybernetics, Silver Spring, Md.) to capture and process images.

FISH analysis of blood cultures from each transgenic kid with probes forthe BC6 transgene showed that all three carry a chromosome 5 transgeneintegration identical to that found in the metaphase plates derived fromthe CFF6 cell line. Moreover, analysis of the least 75 metaphase platesfor each cloned offspring confirmed that they are not mosaic for thechromosome 5 transgenic integration.

As final confirmation that all three kids are derived from thetransgenic CFF6 cell line, PCR-RFLP analysis for the very polymorphicMHC class II DRB gene was undertaken. Typing for the second exon of thecaprine MHC class II DRB gene was performed using PCR-RFLP Typing asdescribed in Amills et al., Immunopathol., 55: 255-260 (1996). Fifteenmicroliters of nested PCR product was digested with 20 units of Rsal(New England Biolabs, Beverly, Mass.). Following digestion, restrictionfragments were separated at room temperature in a 4 to 20% nondenaturingpolycrylamide gel (MVP™ precast gel, Stratagene, La Jolla, Calif.) inthe presence of ethidium bromide.

As illustrated by the RsalI digests of the DRB gene second exon, thethree cloned offspring are identical to each other and identical to theCFF6 donor cell line, whereas the recipient does carry differentalleles.

Induction of Lactation and Transgene Expression of Proteins in Milk

In order to determine whether the targeted mammary gland specificexpression of human ATIII proteins were present in milk, the clonedtransgenic prepubertal clones were transiently induced to lactate. Attwo months of age, the cloned offspring was subjected to a two weekhormonal lactation-induction protocol. Hormonal induction of lactationfor the CFF6-1 female was performed as described in Ryot et al., IndianJ. Anim. Res., 10: 49-51 (1989). The CFF6-1 kid was hand-milked oncedaily to collect milk samples for ATIII expression analysis. All proteinanalysis methods were described in Edmunds et al., Blood, 91: 4561-4571(1998). Concentration of recombinant ATIII in the milk was determined bya rapid reverse-phase HPLC method using a Hewlett Packard 1050 HPLC(Wilmington, Del.) with detection at 214 nm. The ATIII activity wasevaluated by measuring thrombin inhibition with a two-stage colorimetricendpoint assay. Western blot analysis was performed with an affinitypurified sheep anti-ATIII HRP conjugated polyclonal antibody (Sero Tec,Oxford, UK). Samples were boiled for 30 seconds in reducing samplebuffer prior to loading onto a 10-20% gradient gel (Owl Scientific).Electrophoresis was operated at 164 volts (constant) until the dye frontran off the gel.

At the end of the treatment, small milk samples of 0.5 to 10 ml werecollected daily for 20 days. The small initial volumes of milk, 0.5 to 1ml, were typical of the amounts in prepubertal female goats hormonallyinduced to lactate. The volumes increased to 10 ml per day by the timethe female was dried off, 25 days after the onset. The concentration andactivity of ATIII in several of the samples was evaluated. As previouslynoted with does from this specific BC6 transgenic cell line, high levelsof the recombinant ATIII was detected by Western blot analysis (Edmundset al., Blood, 91: 4561-4571 (1998)). The concentration of recombinantATIII in the milk of the cloned offspring was 5.8 grams per liter (205U/ml) at day 5, and 3.7 grams per liter (14.6 U/ml) by day 9. These werein line with levels recorded during the early part of a first naturallactation of does from this BC6 line (3.7 to 4.0 grams per liter).

Healthy transgenic goats were obtained by nuclear transfer of somaticcells to oocytes that were enucleated either in the arrested MetaphaseII or the activated Telophase II-stage. These studies show thatserum-starved cells used to generate term pregnancies are likely toundergo a transition following restoration with 10% serum.

Immunofloresence screening revealed that after 7 days of serumstarvation fetal somatic cells were negative for G₁ stage cyclins D1,D2, D3 and PCNA; whereas within 3 hours of 10% FBS serum-activation amajority (e.g. approximately 70%) expressed these markers.

Reconstruction of an enucleated metaphase II arrested oocyte with thetransfer of a nucleus from a donor karyoplast synchronized at G₀ or G₁of the cell cycle following simultaneous fusion and activation mimic thechronological events occurring during fertilization. The successfuldevelopment to term and birth of a normal and healthy transgenicoffspring following the simultaneous fusion and activation protocol isin contrast with procedures employed in other studies that report therequirement for prolonged exposure of donor nuclei to elevatedcytoplasmic MPF activity to support chromatin remodeling andreprogramming. See Campbell et al. (1996) Nature 380: 64-66; Wilmut etal. (1997) Nature 385: 810-813; Schnieke et al. (1997) Science 278:2130-2133; Cibelli et al. (1998) Science 280: 1256-1258. This resultchallenges the contention that prolonged remodeling of the somaticnuclei in conditions of elevated MPF activity prior to activation isimportant for embryonic and fetal development to term. The results alsodemonstrate that a reconstructed embryo may not have a requirement forprolonged exposure of the donor nucleus to MPF nor are NEBD and PCCentirely requisite events. Rather chromatin remodeling events involvingNEBD and PCC are likely permissive effects of MPF activity and, as such,may not be required for the acquisition of developmental competence ortotipotency. Instead, these events, are likely to serve to facilitatethe acquisition of synchronicity between the cytoplast and thekaryoplast. These events may even be detrimental if normal diploidy isnot maintained when the donor nuclei are induced to undergo PCC withresultant chromosome dispersion due to an aberrant spindle apparatus duein part to MPF activity. Therefore, karyoplast and cytoplastsynchronization with respect to cell cycle is important, first formaintenance of normal ploidy and, second for the proper induction ofgenome reactivation and subsequent acquisition of developmentalcompetence of reconstructed embryos.

Further support is provided in the second method where chromatin-intactmetaphase II arrested oocytes were activated to reduce MPF activity andinduce the oocyte to exit the M-phase and enter the first mitoticcleavage. Approximately 3 hours post-activation, the oocytes wereenucleated at telophase stage prior to the onset of G₁ and fused andsimultaneously activated with a donor karyoplast in G₁ prior to START ofthe cycle. In addition, the simultaneous activation and fusion insuredthat tendencies of non-aged oocytes to revert back to an arrested statewere circumvented. Using this paradigm, a normal and healthy set of twincloned transgenic kids were produced. This procedure inherently providesa homogenous synchronization regimen for the cytoplast to coincidecloser with the donor nuclei in G₁ prior to START. Further preactivationof the oocyte induces a decline in cytoplasmic MPF activity, thusinhibiting the occurrence of NEBD and PCC. These results suggest thatNEBD and PCC is only facultative for the induction of cytoplast andkaryoplast synchrony but necessary for acquisition of proper genomereactivation and subsequent development to term of the nuclear transferembryo using somatic cell nuclei. These finding further suggest thatdifferentiated cells at the G₀ or G₁ stage function similar to embryonicblastomeres with respect to their ability to acquire totipotency whenused in combination with an arrested or an activated recipientcytoplast.

The use of both metaphase II arrested and telophase II cytoplastsprovides dual options for cytoplast preparation in addition to providingan opportunity for a longer time frame to prepare the cytoplast. The useof Telophase II cytoplasts may have several practical and biologicaladvantages. The telophase approach facilitates efficient enucleationavoiding the necessity for chromatin staining and ultravioletlocalization. Moreover, enucleation at telophase enables removal ofminimal cytoplasmic material and selection of a synchronous groups ofactivated donor cytoplasts. This procedure also allows for thepreparation of highly homogenous group of donor nuclei to besynchronized with the cell cycle of the cytoplast. When used for embryoreconstruction, these populations showed a higher rate of embryonicdevelopment in vitro. Thus, reconstructed embryos comprised of asynchronously activated cytoplast and karyoplast are developmentallycompetent.

In addition to a successful transgenic founder production, nucleartransfer of somatic cells allows for the selection of the appropriatetransgenic cell line before the generation of cloned transgenic embryos.This is particularly important in the cases where several proteins areto be co-expressed by the transgenic mammary gland. For example, theavailability of several completely identical transgenic femalesproducing recombinant human ATIII will help determine the extent ofvariation in the carbohydrate structure of this protein, as it isproduced by the mammary gland. Thus, it may be feasible to improve thecharacteristics of the recombinant proteins produces in the transgenicanimal system by varying environmental factors (e.g., nutrition) or toincrease the milk volume yield of lactation-induction protocols todiminish further the time necessary to obtain adequate amounts ofrecombinant proteins for pre-clinical or clinical programs.

The high-level expression of recombinant human ATIII detected in themilk of the CFF6-1 cloned goat illustrates one of the most importantaspects of this technology. By combining nuclear transfer withlactation-induction in prepubetal goats, it may be possible tocharacterize transgenic animals and the proteins they secrete in 8 to 9months from the time of cell line transfection of milk expression. Theamount of milk collected in an induced lactation is not only sufficientto evaluate the recombinant protein yield, but, when mg per mlexpression levels are obtained, is adequate for more qualitativeanalysis (glycosylation, preliminary pharmacokinetics, biological andpharmacological activities). The continued availability of thetransfected donor cell line also insures that genetically identicalanimals can be quickly generated, to rapidly supply therapeutic proteins(with predictable characteristics) for clinical trials.

EXAMPLE 3 Progression of Cytoskeletal and Nuclear Organization During InVitro Maturation of Goat Oocytes

Optimized in vitro maturation of goat oocytes is essential in efforts tocharacterize cell cycle dynamics throughout meiosis in the goat, andparticularly in efforts to promote optimal cytoplasmic and nuclearmaturation for nuclear transfer procedures. Goat oocytes were aspiratedfrom 2-5 mm follicles from stimulated (FSH) and unstimulated(slaughterhouse) animals. Oocyte maturation was assayed by examiningmicrotubule, microfilament and nuclear dynamics in GV (0-2-5 hrs),expected MI (12.5 hrs.), expected MII (22-23 hrs), and aged (44 hrs)oocytes, the maturation of oocytes in two different media was compared:M-199 medium (Earle's salts, 25 mM HEPES), 10% FBS, glutamine (0.1mg/ml); M-199, 10% goat serum (from whole blood and not heatinactivated), and glutamine (0.1 mg/ml). Oocytes were simultaneouslyfixed and extracted suing a cytoskeletal stabilizing buffer (MTSB) for 1hour at 37° C., and then washed and stored in block solution (PBS-Azide.0.2% powdered milk 2% normal goat serum, 1% BSA, 0.1 M Glycine 0.1%Triton X-100) at 4° C. prior to analysis. Oocytes were processed forimmunofluorescence localization of microtubules, microfilaments andchromatin using anti-α and β tubulin monoclonal antibodies, Oregon Greenphalloidin (Molecular Probes) and Hoechst 33258, respectively.Fluorescence signal was visualized using both standardimmunoflourescence and confocal microscopy.

Preliminary estimates of oocytes matured in the presence of FBS showthat approximately 90% progressed improperly (N=40 to date). MI spindleformation was compromised with a subsequence lack of polar bodyextrusion. Those oocytes which progressed to MII did so withoutextruding the first polar body and the MII spindle was improperly placedin the center of the cell instead of near the cortex. Preliminaryestimates of oocytes matured in the presence of goat serum show thatapproximately 90% progressed through meiosis (N=480 to date). Themorphology of the cortically placed microfilament network did not varysignificantly between oocytes matured in FBS and goat serum. There maybe come possible defect in polar body extrusion dynamics which is maskedby the failure of the improper MI spindle morphogenesis. Further, therewas no clear difference in meiotic maturation in terms of thecytoskeleton between oocytes collected from stimulated and unstimulatedanimals.

These data indicate that FBS is not sufficient for normal meioticprogression in vitro in the goat. Maturation media containing goat serumsupports morphologically normal microtubule and spindle organization anddynamics as well as expected nuclear meiotic hallmarks. Hallmarks suchas the development of the rim around the GV nucleus that is indicativeof acquisition of meiotic competence as well as the condensation of DNArequisite for MI and MII progression. The goat serum which was not heatinactivated, while not selected specifically from male or femaleanimals, must be sufficiently enriched with hormones, gonadotrophins andother factors to support meiotic progression in goat oocytes.

EXAMPLE 4 Murine Cloning

The objective of this experiment was to produce nuclear transfer embryosusing activated Telophase II mouse oocytes combined with somatic cells(cumulus cells).

Oocytes were collected from superovulated mice by flushing oviducts tocollect metaphase II stage oocytes. Oocytes were then activated by oneof two methods to reach the Telophase II state in vitro: culture-inducedCa²⁺ activation or ionomycin activation (4 μM, 5 min), and enucleated.Karyoplasts were prepared from cumulus cells (natural G₀ stage) andnuclear transfer was conducted followed by electrical fusion withTelophase II cytoplasts.

The results of these experiments as shown in Table 2. TABLE 2Development of Mouse Nuclear Transfer Embryos using activated TelophaseII protocol (3 replicate experiments) Protocol Number Percent OocytesRecovered 202 Activation: 47/52 90 Culture induced Ca²⁺ Activation:46/50 92 Ionomycin induced Ca²⁺ Oocytes Enucleated &  85 42Reconstructed Embryo cleavage (2-cell)  51 60 Transferred embryos  21 41Recipients  3 Na Pregnancy (day 11-15) 2/3 66 Term Pregnancy  1 33(ceasarian day 20) Offspring (total)  3 na (stillborn) Offspring (% oftotal  3/21 14.3 transferred)

EXAMPLE 5 In Vitro Maturation of Transported Pig Oocytes in a DefinedProtein-Free Medium

Improved efficiency of in vitro embryo production of embryo cloningusing nuclear transfer, will rely on a detailed understanding ofcellular and nuclear progression during in vitro oocyte maturation (IVM)in pigs. The objective of this study was to demonstrate precisely theprogression of chromatin and microtubule dynamics in pig oocytes duringIVM using a defined protein-free medium. Follicles from abattoir ovarieswere aspirated with 18 G-needles. Cumulus-oocyte-complexes (COCs) werewashed and cultured in 0.5 mL tubes in pre-equilibrated (5% CO₂) M199medium containing polyvinyl alcohol (0.1%) cystein (0.1 mg/mL), EGF (10ng/mL), Pen/Strep, hCG (10 IUmL) and PMSG (10 IU/mL; Abeydeera et al.Biol. Reprod. 58: 1316-1320 (1998). Oocytes were then transported(Minitube incubator, 39° C.) over night by air-courier. Upon receipt (24h of maturation COGs were freed of cumulus and corona cells and fixed inmicrotubule stabilization buffer (1 h, 37° C.). Fixed oocytes werestored (PBS, 0.1% PVA) at 4° C. prior to staining. Fluorescence stainingfor microtubules and chromatin was preformed. Stained oocytes weremounted on slides (PBS/50% glycerine) and evaluated at ×400magnification (see Table 3). TABLE 3 Developmental stages of pig oocytesat different time-points of in vitro maturation Time of germinalMaturation Germinal vesicle anaphase/ degenerated or (h post Vesiclebreakdown MI telophase MII spontaneously aspiration) N GV (%) GVBD (%)(%) (%) (%) activated (%) 26 h 53 16 (30.2) 23 (43.4) 11 (20.8) 1 (1.9) 0 2 (3.8) 29 h 51 18 (35.3) 12 (23.5) 17 (33.3) 1 (2.0)  1 (2.0) 2(3.9) 32 h 53  5 (9.4) 10 (18.9) 27 (50.9) 3 (5.7)  2 (3.8) 6 (11.3) 35h 52  3 (5.8)  7 (13.5) 29 (55.8) 6 (11.5)  5 (9.6) 2 (3.8) 38 h 54  6(11.1)  3 (5.6) 25 (46.3) 5 (9.3) 14 (25.9) 1 (1.9) 41 h 51  4 (7.8)  6(11.8)  8 (15.7) 2 (3.9) 27 (52.9) 4 (7.8) 44 h 99 10 (10.1)  4 (4.0)  8(8.1) 0 76 (76.8) 1 (1.0)

Progressive changes in chromatin and microtubular configuration, fromasters that associated to the chromatin during prometaphase, to furtherchromatin condensation and elongation of the meiotic spindle untilTelophase I, and followed by the extrusion of the first polar body wereobserved and photodocumented. In conclusion, the percentage ofMII-oocytes at 44 h of maturation (76%) is comparable to the resultsafter routine IVM using the same protocol (86% Abeydeera et al., 1998).Our data indicate, that the defined, protein-free medium effectivelysupports IVM of procine oocytes, subjected to overnight transport duringthe first 24 h of IVM.

EXAMPLE 6 Differential Expression of E-Cadherin and Na+/K+ ATPaseProteins in Parthenogenetic Pig Embryos

Parthenogenetic activation of porcine oocytes supports limited andvariable embryonic development in vitro and in vivo. As parthenotesdevelop devoid of parental genes, the objective of the study was toassess the ability of the maternal genome to direct the keydevelopmental processes of embryo compaction and cavitation. Expressionand localization of E-cadherin, an important transmembrane cell adhesionmolecule involved in compaction, and Na+/K=ATPase, an activetransmembrane ion transporting enzyme involved in cavitation, werecharacterized in both in vivo produced embryos and parthenogenetic pigembryos that development in vitro following activation. Pig ovaries wereobtained from slaughter house material and oocytes were aspirated (2.6mm follicles), washed in Hepes-buffered Tyrode's solution (HbT) andmatured in Waymouth MB medium containing 10 IU hCG, 10% fetal bovineserum and 10% (v/v) porcine follicular fluid for 20 h, followed by anadditional 24 h without hormones. Oocytes were activated by electricalpulse (5 V AC followed by 1.44 kV/cm DC for 31.2 msec) in Hbt-0.3 Mmannitol in 5% HbT, and transferred surgically into ligated oviducts ofsynchronous recipient gilts. Parthenogenetic and in vivo produced(natural cycle) embryos were recovered at cleavage or morula stages ofdevelopment on days 3 or 6 following transfer. Embryos were washed(2×HbT) and fixed in either microtubule stabilizing buffer (MTSB; 0.1 MPIPES, 5 mM MgCl₂, 2.5 mM EGTA, 0.01% aprotinin, 1 mM DTT, 50% deuteriumoxide, 1 μM Taxol®, 0.1% Triton X-100, 3.7% formalin) or 4%paraformaldehyde/0.5% saponin for 60 minutes at 37° C. Individualembryos were processed for immunofluorescence localization of E-cadherin(anti-E-cadherin MAB/GAR-Cy3, 23° C., 2 hr) or Na+/K+ ATPase (anti-α/βsubunit MAB/GAR-Cy3, 23° C. 2 h), followed by staining of nuclei(Hoechst 33258, 10 μg/ml in PBS, 37° C. 15 min.). Single embryos weremounted on glass slides (50% glycerol in PBS) and subsequently analyzedusing conventional and confocal fluorescence microscopy. Na+/K+ ATPasestaining was first apparent at abutting blastomere cell borders ofcompacted morulae in both in vivo and parthenogenetic embryos.Similarly, Na+ pump subunits were uniformly localized at the basallateral domains of trophectoderm cells of both in vivo andparthenogenetic blastocysts. E-cadherin was localized to corticalregions of cell-cell contact in most blastomeres of 8-cell embryos anduncompacted and compacted morulae and in trophectodermal cells ofblastocysts produced in vivo. In contrast, E-cadherin was observed asheterogeneous cortical staining among blastomeres of parthenogeneticembryos at all embryonic stages. Further, parthenogenetic embryos ofpoor morphological quality displayed extensive disruption of corticalE-cadherin staining. These results suggest that expression of E-cadherin(compaction) but not Na+/K+ ATPase (cavitation) may require paternalgenome participation for normal blastomere differentiation prior tocompaction since differential expression of these proteins coincidestemporally with the time of zygotic gene activation in the pig.Alternatively, the observed heterogeneous protein/gene expression andcellular localization patterns may be a result of aberrant chromosomalsegregation.

The teachings of all the patents, patent applications and publicationscited herein are incorporated by reference in their entirety.

Equivalents

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1-30. (canceled)
 31. A method of enucleating an oocyte having a meioticspindle apparatus, comprising exposing the oocyte with at least onecompound that destabilizes the meiotic spindle apparatus.
 32. The methodof claim 31, wherein destabilizing the meiotic spindle apparatus furtherincludes destabilizing microtubules, chromosomes, or centrioles.
 33. Themethod of claim 31, wherein the compound is demecolcine, nocodazole,colchicine, or paclitaxel.
 34. The method of claim 31, wherein furtherincluding altering the temperature, osmolality or composition of mediumwhich surrounds the oocyte.
 35. A method of preparing an oocyte fornuclear transfer, wherein the oocyte has a meiotic spindle apparatus,comprising the steps of: a. exposing the oocyte to ethanol, ionophore,and/or to electrical stimulation, to thereby obtain an activated oocyte,and b. subjecting the activated oocyte to at least one compound thatdestabilizes the meiotic spindle apparatus, to thereby enucleate theactivated oocyte.
 36. The method of claim 35, wherein destabilizing themeiotic spindles apparatus includes destabilizing microtubules,chromosomes, or centrioles.
 37. The method of claim 35, wherein thecompound is demecolcine, nocodazole, colchicine, or paclitaxel.
 38. Themethod of claim 35, wherein the activated oocyte is in a stage of ameiotic cell cycle selected from the group consisting of: metaphase I,anaphase I, anaphase II and telophase II.